Fig. 4

Flow chart describing the in vivo experimental plan. Day 35 spleens were harvested and a limiting dilution assay (LDA) set-up. Condition 1: the medium used in the LDA only contained antibiotics selecting for single NMT or double NMT knockouts, depending on the mutants initially injected, in order to determine the total parasite burden. Conditions 2–4 were set up to determine the individual parasite burden, i.e. which plasmid are the parasite still carrying: Condition 2: LDA medium contained also nourseothricin to select for the NMT-TK plasmid. Condition 3: LDA medium contained also G418 to select for the NMT-tdtom or the tdtom plasmid. Condition 4: the medium contained also nourseothricin and G418 to select either for the NMT-TK and NMT-tdtom plasmid or the NMT-TK and tdtom plasmid